Two of the most common types of osteoporosis are postmenopausal and senile osteoporosis. Osteoporosis affects both men and women, and, taken with other abnormalities of bone, presents an ever-increasing health risk for an aging population. The most common type of osteoporosis is that associated with menopause. Most women lose between 20-60% of the bone mass in the trabecular compartment of the bone within 3-6 years after the cessation of menses. This rapid bone loss is generally associated with an increase of bone resorption and formation. However, the resorptive cycle is more dominant and the result is a net loss of bone mass. Osteoporosis is a common and serious disease among postmenopausal women. There are an estimated 25 million women in the United States alone who are afflicted with this disease. The results of osteoporosis are personally harmful, and also account for a large economic loss due to its chronicity and the need for extensive and long-term support (e.g., hospitalization and nursing home care) from disease sequelae. This is especially true in elderly patients. Additionally, while osteoporosis is generally not thought of as a life-threatening condition, a 20-30% mortality rate is related to hip fractures in elderly women. A large percentage of this mortality rate can be directly associated with postmenopausal osteoporosis.
The most vulnerable tissue in the bone to the effects of postmenopausal osteoporosis is the trabecular bone. This tissue is often referred to as spongy bone and is particularly concentrated near the ends of the bone, near the joints, and in the vertebrae of the spine. The trabecular tissue is characterized by small structures which inter-connect with each other as well as the more solid and dense cortical tissue which makes up the outer surface and central shaft of the bone. This cris-cross network of trabeculae gives lateral support to the outer cortical structure and is critical to the biomechanical strength of the overall structure. In postmenopausal osteoporosis, it is primarily the net resorption and loss of the trabeculae which lead to the failure and fracture of the bone. In light of the loss of the trabeculae in postmenopausal women, it is not surprising that the most common fractures are those associated with bones which are highly dependent on trabecular support, e.g., the vertebrae, the neck of the femur, and the forearm. Indeed, hip fracture, Colle's fractures, and vertebral crush fractures are indicative of postmenopausal osteoporosis. Osteoporosis affects cortical as well as trabecular bone. Alterations in endosteal bone resorption and Haversian remodeling with age affect cortical thickness and structural integrity contributing the increased risk for fracture.
One of the earliest generally accepted methods for treatment of postmenopausal osteoporosis was estrogen replacement therapy. Although this therapy frequently is successful, patient compliance is low, primarily due to the undesirable side-effects of chronic estrogen treatment. Frequently cited side-effects of estrogen replacement therapy include reinitiation of menses, bloating, depression, and, potentially, increased risk of breast or uterine cancer. In order to limit the known threat of uterine cancer in women who have not had a hysterectomy, a protocol of estrogen and progestin cyclic therapy is often employed. This protocol is similar to that used in birth control regimens, and often is not tolerated by women because of the side-effects characteristic of progestin. More recently, certain antiestrogens, originally developed for the treatment of breast cancer, have been shown in experimental models of postmenopausal osteoporosis to be efficacious. Among these agents is raloxifene (See, U.S. Pat. No. 5,393,763; Black et. al., J. Clin. Invest., 93:63-69 (1994); and Ettinger et al., JAMA 282:637-45 (1999)). In addition, tamoxifen, a widely used clinical agent for treating breast cancer, has been shown to increase bone mineral density in post menopausal women suffering from breast cancer (Love et al., N. Engl. J. Med., 326:852-856 (1992)).
Another therapy for the treatment of postmenopausal osteoporosis is the use of calcitonin. Calcitonin is a naturally occurring peptide which inhibits bone resorption and has been approved for this use in many countries (Overgaard et al., Br. Med. J., 305:556-561 (1992)). The use of calcitonin has been somewhat limited, however. Its effects are very modest in increasing bone mineral density, and the treatment is very expensive. Another therapy for the treatment of postmenopausal osteoporosis is the use of bisphosphonates. These compounds were originally developed for treating Paget's disease and malignant hypercalcemia. They have been shown to inhibit bone resorption. Alendronate, a bisphosphonate, has been approved for the treatment of postmenopausal osteoporosis. These agents may be helpful in the treatment of osteoporosis, but these agents also have potential liabilities which include osteomalacia, extremely long half-life in bone (greater than 2 years),; and possible “frozen bone syndrome,” e.g., the cessation of normal bone remodeling.
Senile osteoporosis is similar to postmenopausal osteoporosis in that it is marked by the loss of bone mineral density and resulting increase in fracture rate, morbidity, and associated mortality. Generally, it occurs in later life, i.e., after 70 years of age. Historically, senile osteoporosis has been more common in females, but with the advent of a more elderly male population, this disease is becoming a major factor in the health of both sexes. It is not clear what, if any, role hormones such as testosterone or estrogen have in this disease, and its etiology remains obscure. Treatment of this disease has not been very satisfactory. Hormone therapy, estrogen in women and testosterone in men, has shown equivocal results; calcitonin and bisphosphonates may be of some utility.
The peak mass of the skeleton at maturity is largely under genetic control. Twin studies have shown that the variance in bone mass between adult monozygotic twins is smaller than between dizygotic twins (Slemenda et al., J. Bone Miner. Res., 6: 561-567 (1991); Young et al., J. Bone Miner. Res., 6:561-567 (1995); Pocock et al., J. Clin. Invest., 80:706-710 (1987); Kelly et al., J. Bone Miner. Res., 8:11-17 (1993)). It has been estimated that up to 60% or more of the variance in skeletal mass is inherited (Krall et al., J. Bone Miner. Res., 10:S367 (1993)). Peak skeletal mass is the most powerful determinant of bone mass in elderly years (Hui et al., Ann. Int. Med., 111:355-361 (1989)), even though the rate of age-related bone loss in adult and later life is also a strong determinant (Hui et al., Osteoporosis Int., 1:30-34 (1995)). Since bone mass is the principal measurable determinant of fracture risk, the inherited peak skeletal mass achieved at maturity is an important determinant of an individual's risk of fracture later in life. Thus, study of the genetic basis of bone mass is of considerable interest in the etiology of fractures due to osteoporosis.
Recently, a strong interest in the genetic control of peak bone mass has developed in the field of osteoporosis. The interest has focused mainly on candidate genes with suitable polymorphisms to test for association with variation in bone mass within the normal range, or has focused on examination of genes and gene loci associated with low bone mass in the range found in patients with osteoporosis. The vitamin D receptor locus (VDR) (Morrison et al., Nature, 367:284-287 (1994)), PTH gene (Howard et al., J. Clin. Endocrinol. Metab., 80:2800-2805 (1995); Johnson et al., J. Bone Miner. Res., 8:11-17 (1995); Gong et al., J. Bone Miner. Res., 10: S462 (1995)) and the estrogen receptor gene (Hosoi et al., J. Bone Miner. Res., 10: S170 (1995); Morrison et al., Nature, 367:284-287 (1994)) have figured most prominently in this work. These studies are difficult because bone mass (i.e, the phenotype) is a continuous, quantitative, polygenic trait, and is confounded by environmental factors such as nutrition, co-norbid disease, age, physical activity, and other factors. Also, this type of study design requires large numbers of subjects. In particular, the results of VDR studies to date have been confusing and contradictory (Garnero et al., J. Bone Miner. Res., 10:1283-1288 (1995); Eisman et al., J. Bone. Miner. Res., 10:1289-1293 (1995); Peacock, J. Bone Miner. Res., 10:1294-1297 (1995)). Furthermore, thus far, the art has not determined the mechanism(s) whereby the genetic influences exert their effect on bone mass.
While it is well known that peak bone mass is largely determined by genetic rather than environmental factors, studies to determine the gene loci (and ultimately the genes) linked to variation in bone mass are difficult and expensive. Study designs which utilize the power of linkage analysis, e.g., sib-pair or extended family, are generally more informative than simple association studies, although the latter do have value. However, genetic linkage studies involving bone mass are hampered by two major problems. The first problem is the phenotype, as discussed briefly above. Bone mass is a continuous, quantitative trait, and establishing a discrete phenotype is difficult. Each anatomical site for measurement may be influenced by several genes, many of which may be different from site to site. The second problem is the age component of the phenotype. By the time an individual can be identified as having low bone mass, there is a high probability that their parents or other members of prior generations will be deceased and therefore unavailable for study, and younger generations may not have even reached peak bone mass, making their phenotyping uncertain for genetic analysis.
Thus, there is a need in the art for additional research tools for the elucidation of the rholecular mechanism of bone modulation, for the screening and development of candidate drugs, and for treatments of bone development and bone loss disorders. The present invention is directed to these, as well as other, important ends.
In addition to bone modulation, the present invention relates to modulation of lipid levels. Cardiovascular disease is the most common cause of mortality in the United States, and atherosclerosis is the major cause of heart disease and stroke. It is widely appreciated that cholesterol plays an important role in atherogenesis. Normally, most cholesterol serves as a structural element in the walls of cells, whereas much of the rest is in transit through the blood or functions as the starting material for the synthesis of bile acids in the liver, steroid hormones in endocrine cells and vitamin D in skin. The transport of cholesterol and other lipids through the circulatory system is facilitated by their packaging into lipoprotein carriers. These spherical particles comprise protein and phospholipid shells surrounding a core of neutral lipid, including unesterified (“free”) or esterified cholesterol and triglycerides.
Risk for atherosclerosis increases with increasing concentrations of low density lipoprotein (LDL) cholesterol, whereas risk is inversely proportional to levels of high-density lipoprotein (HDL) cholesterol. The receptor-mediated control of plasma LDL levels has been well-defined, and recent studies have now provided new insights into HDL metabolism.
The elucidation of LDL metabolism began in 1974 by Michael Brown and Joseph Goldstein. In brief, the liver synthesizes a precursor lipoprotein (very low density lipoprotein, VLDL) that is converted during circulation to intermediate density lipoprotein (IDL) and then to LDL. The majority of the LDL receptors expressed in the body are on the surfaces of liver cells, although virtually all other tissues (“peripheral tissues”) express some LDL receptors. After binding, the receptor-lipoprotein complex is internalized by the cells via coated pits and vesicles, and the entire LDL particle is delivered to lysosomes, wherein it is dissembled by enzymatic hydrolysis, releasing cholesterol for subsequent cellular metabolism. This whole-particle uptake pathway is called “receptor-mediated endocytosis.” Cholesterol-mediated feedback regulation of both the levels of LDL receptors and cellular cholesterol biosynthesis help ensure cellular cholesterol homeostasis. Genetic defects in the LDL receptor in humans results in familial hypercholesterolemia, a disease characterized by elevated plasma LDL cholesterol and premature atherosclerosis and heart attacks. One hypothesis for the deleterious effects of excess plasma LDL cholesterol is that LDL enters the artery wall, is chemically modified, and then is recognized by a special class of receptors called macrophage scavenger receptors, that mediate the cellular accumulation of the LDL cholesterol in the artery, eventually leading to the formation of an atherosclerotic lesion.
The major lipoprotein classes include intestinally derived chylomicrons that transport dietary fats and cholesterol, hepatic-derived VLDL, IDL, and LDL that can be atherogenic, and hepatic- and intestinally-derived HDL that are antiatherogenic. Apoprotein B (ApoB) is necessary for the secretion of chylomicrons (ApoB48) and VLDL, IDL, and LDL (ApoB100). Plasma levels of VLDL triglycerides are determined mainly by the rates of secretion in LDL lipolytic activity. Plasma levels of LDL cholesterol are determined mainly by the secretion of ApoB100 into plasma, the efficacy with which VLDL are converted to LDL and by LDL receptor-mediated clearance. Regulation of HDL cholesterol levels is complex and is affected by rates of synthesis of its Apo proteins, rates of esterification of free cholesterol to cholesterol ester by LCAT, levels of triglyceride-rich lipoproteins and CETP-mediated transfer of cholesterol esters from HDL, and clearance from plasma of HDL lipids and Apo proteins.
Normal lipoprotein transport is associated with low levels of triglycerides and LDL cholesterol and high levels of HDL cholesterol. When lipoprotein transport is abnormal, lipoprotein levels can change in ways that predispose individuals to atherosclerosis and arteriosclerosis (see Ginsburg, Endocrinol. Metab. Clin. North Am., 27:503-19 (1998)).
Several lipoprotein receptors may be involved in cellular lipid uptake. These receptors include: scavenger receptors; LDL receptor-related protein/α2-macroglobulin receptor (LRP); LDL receptor; and VLDL receptor. With the exception of the LDL receptor, all of these receptors are expressed in atherosclerotic lesions while scavenger receptors are mostly expressed in macrophages, the LRP and VLDL receptors may play an important role in mediating lipid uptake in smooth muscle cells (Hiltunen et al., Atherosclerosis, 137 suppl.:S81-8 (1998)).
A major breakthrough in the pharmacologic treatment of hypercholesterolemia has been the development of the “statin” class of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG CoA reductase) inhibitory drugs. 3-hydroxy-3-methylglutaryl-CoA reductase is the rate controlling enzyme in cholesterol biosynthesis, and its inhibition in the liver stimulates LDL receptor expression. As a consequence, both plasma LDL cholesterol levels and the risk for atherosclerosis decrease. The discovery and analysis of the LDL receptor system has had a profound impact on cell biology, physiology, and medicine.
HDL is thought to remove unesterified, or “free” cholesterol (FC) from peripheral tissues, after which most of the cholesterol is converted to cholesterol ester (CE) by enzymes in the plasma. Subsequently, HDL cholesterol is efficiently delivered directly to the liver and steroidogenic tissues via; a selective uptake pathway and the HDL receptor, SR-BI (class B type I scavenger receptor) or, in some species, transferred to other lipoproteins for additional transport in metabolism (see Krieger, Proc. Natl. Acad. Sci. USA, 95:4077-4080 (1998)).
These issues illustrate a need in the art for additional research tools for the elucidation of the molecular mechanism of lipid modulation, for the screening and development of candidate drugs, and for treatments of lipid levels and lipid level modulation disorders. The present invention is directed to these, as well as other, important ends.